Key Words: SurePath. CINtec; p16; Ki-67, MIB1; sensitivity; specificity;

Transcription

1 Evaluation of CINtec PLUS 1 Testing as an Adjunctive Test in ASC-US Diagnosed Surepath 1 Preparations Neil Edgerton, M.D., Cynthia Cohen, M.D., andmomint. Siddiqui,M.D., F.I.A.C.* The CINtec PLUS 1 system is an immunohistochemical cocktail composed of antibodies against p16 INK4a (surrogate of HPV infection) and Ki-67 (proliferation marker) meant to improve the sensitivity and specificity for detecting high-grade dysplasia (HGD). In the presence of dysplasia, a red chromogen marks Ki-67 expression in the nucleus and a brown chromogen marks cytoplasmic p16 INK4a expression. Only cells showing dual staining are interpreted as positive. This retrospective study examined the performance of CINtec PLUS testing when performed on ASC-US diagnosed samples. Comparison was made to highrisk HPV DNA test results and colposcopic biopsy results. Technical considerations in the interpretation of this immunohistochemical stain are additionally discussed. CINtec PLUS showed modest sensitivity (64%) and specificity (53%) in identifying the presence of HGD at surgical biopsy. Positive and negative predictive values for HGD were 28% and 83%, respectively. HR- HPV DNA test yielded sensitivity of 100% and specificity of 21%. During interpretation, squamous metaplasia and endocervical cells were seen to show individual staining for p16 INK4a or Ki-67. Individual staining, when present within three dimensional cellular groups common to SurePath 1 preparations, can be time-intensive to interpret necessitating thoughtful examination at high power. The Pap test with HR-HPV DNA testing is a highly sensitive test. A specific test is needed to prevent false positives and over treatment. The CINtec 1 system provides a modest increase in specificity beyond HR-HPV DNA testing. Future study of its appropriateness and cost-ffectiveness in a treatment algorithm are warranted. Diagn. Cytopathol. 2013;41: ' 2011 Wiley Periodicals, Inc. Key Words: SurePath CINtec; p16; Ki-67, MIB1; sensitivity; specificity; Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia *Correspondence to: Momin T. Siddiqui, M.D., F.I.A.C., Department of Pathology, Room G179B, Emory University Hospital, 1364 Clifton Rd. NE, Atlanta, GA mtsiddi@emory.edu Received 21 March 2011; Accepted 11 May 2011 DOI /dc Published online 27 June 2011 in Wiley Online Library (wileyonlinelibrary.com). The 2001 Bethesda Conference standardized diagnostic terminology within the field of cervical cytopathology. 1 This standardization, in turn, allowed for well developed treatment algorithms to detect and treat cervical dysplasia and carcinoma. 2 Where cervical cancer was once the most common carcinoma to affect women, it now numbers thirteenth in the United States. 3 In 1950, the rate of cervical cancer within the US was 26.2 per 100, By 2006, the rate had fallen to 5.2 per 100, The widespread use of Pap testing is a public health success. As proof of this in 2008, nearly 88% of women aged had received an annual cervical cancer screening thus demonstrating the acceptance of this test by practitioner and patient alike. 5 The broad adoption of HR-HPV testing has furthered the role of the Pap test. The use of this ancillary test on residual sampled material allows for the extremely high sensitivity expected of a screening test. Further, HPV testing provides another means of stratifying risk within the treatment algorithms. 2,6 Given such success, efforts are now turning to increasing the specificity of the Pap test. Effort is being directed at identifying the subset of women who harbor high-grade dysplasia (HGD) within those Pap tests diagnosed as atypical squamous cells of undetermined significance (ASCUS) or low grade squamous intraepithelial lesion (LSIL). Human papilloma virus drives dysplastic changes that ultimately lead to carcinoma. The progression of disease is long and numbers in years. 7 In the majority of cases, viral infection is transient in nature. 8,9 Only a small set of patients progress to higher grades of dysplasia. Some of these patients will still undergo spontaneous regression of dysplasia without the development of carcinoma. 10 Carcinoma represents an accumulation of insults to the cervical squamous mucosa in which the HPV virus has integrated into the host DNA due to the inability of the immune system to clear the virus. This specific progression occurs via the direct action of two viral oncoproteins E6 and E7. ' 2011 WILEY PERIODICALS, INC. Diagnostic Cytopathology, Vol 41, No 1 35

2 EDGERTON ET AL. The oncoprotein E6 interacts with host p53 tumor suppressor protein resulting in degradation of p53 thus preventing apoptotic death of the cell. 11 The oncoprotein E7 interacts with host retinoblastoma protein, such that when Rb protein is bound, it is unavailable to halt the cell cycle. 12 Ultimately, the disabling of these two proteins allows for unhindered cellular proliferation. p16 INK4a, is a cyclin-dependent kinase inhibitor, that serves to negatively regulate cellular proliferation. 13 Its expression is upregulated in viral integration; thus, immunohistochemical staining for p16 INK4a is a surrogate marker of HPV infection. Active cellular proliferation is identified by nuclear expression of Ki-67. Ki-67 is an antigen that is present during cellular proliferation, specifically late G1 and S phases. 14 The CINtec PLUS 1 system is an immunohistochemical cocktail composed of antibodies against p16 INK4a and Ki-67 meant to improve the detection of HGD. Specifically, it has been examined as an adjunctive test that would serve to increase overall specificity. 15 In the presence of dysplasia, a red chromogen marks Ki-67 expression in the nucleus and a brown chromogen marks cytoplasmic p16 INK4a expression. This retrospective study examined the performance characteristics of CINtec PLUS testing when performed on ASC- US diagnosed samples prepared in SurePath preparations. Materials and Methods Patients and specimens A total of 63 SurePath 1 Pap test slides diagnosed as ASC-US with correlative colposcopic biopsy were identified over a 17 month period from Emory University Hospital. Correlative colposcopic biopsy diagnoses were derived from the medical record. Follow-up biopsy results represent biopsies diagnosed by the routine staff physicians within our institution, and were secondarily reviewed as part of the institution s QA/QC program. In the event multiple biopsies were taken, the most severe dysplasia (highest CIN) was recorded. Digene Hybrid Capture 1 II HPV DNA test (Digene Corporation, Gaithersburg, MD) was performed on Sure- Path specimens at ARUP Laboratories (Salt Lake City, Utah). The samples were received within one day of collection and stored at room temperature until testing was performed within 2 week of specimen collection. Digene Hybrid Capture 1 II HPV DNA testing was performed according to the manufacturer s recommendations using the high-risk probes with the positive and negative control samples run in triplicate and result validation using HC2 software, version 2.0. The high-risk HPV panel consisted of 13 types of HPV (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). The hybridized product is detected by chemiluminescent reporter system. The test results are given as relative light unit ratios. The manufacturer has set a positive cut-off value of 1 pg HPV DNA per ml in the specimen. Immunohistochemistry was performed on previously prepared SurePath Papanicolaou stained slides (Becton, Dickinson and Company, Franklin Lakes, NJ), which were destained according to manufacturer provided protocol. SurePath slides were incubated overnight in xylene to remove the coverslips. A secondary wash with fresh xylene removed residual mounting medium. Progressive rehydration from ethanol to deionized water was performed. Antigen retrieval was performed with the proprietary Epitope Retrieval Solution provided within the CINtec Cytology Kit (mtm Laboratories, Heidelberg, Germany). Retrieval was carried out within a water bath where the temperature was raised from room temperature to C within 40 minutes. Once at 958C, the slides were incubated for 15 minutes. Slides were cooled for 20 minutes within the Epitope Retrieval Solution. Slides were then placed in wash buffer (Tris-buffered saline) for 5 minutes prior to immunostaining. All slides were loaded on an automated system (Dako AutoStainer plus, Carpinteria, CA) and incubated with primary antibodies per the provided CINtec PLUS Kit protocol. Slides were then counterstained with hematoxylin for 5 minutes. The slides were then air-dried and 40 ll of CINtec PLUS Mount aqueous mounting medium was swirled over the specimen. The aqueous mounting medium was allowed to dry before placing slides in xylene for 1 minute, and manual coverslipping with xylene based mounting medium. A positive control was derived from a SurePath slide diagnosed as HSIL and confirmed CIN III by biopsy. A negative control was derived from a SurePath slide diagnosed as negative for intraepithelial lesion or malignancy. Analysis Slides were analyzed by a single pathologist without adjudication. The pathologist knew the sample was composed of ASC-US diagnosed cytology specimens, but was blinded to the specific cytology diagnosis, HR-HPV results, and surgical biopsy findings. The slides were interpreted as positive or negative for CINtec PLUS staining based on manufacturer provided atlas and guidelines. In the presence of dysplasia, a red chromogen marked Ki-67 expression in the nucleus and a brown chromogen marked cytoplasmic p16 INK4a expression. Classically, immunohistochemical staining for p16 INK4a is nuclear and cytoplasmic. In this double stain, p16 INK4a nuclear staining is obscured by the intense red chromogen that marks Ki-67. The testing was considered positive if a minimum of one cell showed simultaneous dual staining (Figs. 1a and b). The utility of CINtec staining on SurePath slides was determined using sensitivity and specificity calcula- 36 Diagnostic Cytopathology, Vol 41, No 1

3 CINtec PLUS SENSITIVITY AND SPECIFICITY Fig. 1. a: Dysplastic cell interpreted as positive for CINtec staining exhibits nuclear Ki-67 (red chromogen) and cytoplasmic p16 (brown chromogen), 340. b: Cell on top with cytoplasmic and nuclear p16 interpreted as negative. Cell on bottom interpreted as positive, exhibits nuclear Ki-67 and cytoplasmic p16, 360. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Table I. Correlative Results Biopsy Cytology NILM CIN I CINII CIN III ASC-US (n ¼ 3) tions performed by the JavaSTAT online contingency table calculator. Results Within our sample set, 44% of atypical squamous cell diagnoses were found to have a CIN lesion (CIN I III) (Table I). Fourteen cases (22%) of our sample set of ASC-US cases had HGD (CIN II/III) at the time of cervical biopsy. No cases of cervical carcinoma were present. Staining with the CINtec PLUS system has modest sensitivity (64%) and specificity (53%) in identifying the presence of HGD at surgical biopsy (Table II). Positive and negative predictive values for HGD were 28% and 84%, respectively (Table II). HR-HPV DNA testing yielded a sensitivity of 100% and specificity of 21% (Table III). The data for HR-HPV testing are derived from a small sample, but do show it to be a sensitive test for disease exclusion. Results of CINtec Plus interpretation on Pap slides were sorted by diagnosis at the time of colposcopic biopsy (Table IV). Since the ultimate goal of this test is risk stratification, we assume that the gains in specificity are achieved by demonstrating morphologic changes seen within the Pap test as the result of HPV infection (proved by p16 and Ki-67 expression), and that these changes are driven by the presence of HPV (proved by HR-HPV DNA testing). These two independent tests are thus interpreted in series, whereby both tests must be positive to give a higher level of risk. This performance of two tests in series increases Table II. CINtec PLUS Staining Sensitivity and Specificity in Detecting HSIL (CIN II/III) Sensitivity 64% Specificity 53% PPV 28% NPV 84% PPV, positive predictive value; NPV, negative predictive value. Table III. HR-HPVDNA Testing Sensitivity and Specificity in Detecting HSIL (CIN II/III) Sensitivity 100% Specificity 21% PPV 50% NPV 100% PPV, positive predictive value; NPV, negative predictive value. Table IV. CINtec PLUS 1 Stain Interpretation Within Pap Slides Compared by Final Biopsy Diagnosis specificity. The overall specificity or net specificity of these two tests is computed by summing the specificity of the two tests minus the product of the two test s specificity (A+B) (A 3 B). The overall specificity, in this Diagnostic Cytopathology, Vol 41, No 1 37

4 EDGERTON ET AL. Fig. 2. Squamous metaplasia recognized by sharp cytoplasmic border, exhibiting diffuse p16 staining (brown chromogen), 340. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] study, of interpreting a case that stained positive with the CINtec PLUS system and was also HR-HPV DNA positive was found to be 63%. Discussion In this study, we document our experience with the CINtec PLUS immunohistochemical protocol. Knowing that significant interobserver variability exists in the atypical squamous cell categories, we explored the performance of the CINtec PLUS immunohistochemical staining protocol and its ability to identify HGD. The value of p16 in the analysis of cervical biopsy has been reliably demonstrated. Evaluation with p16 immunohistochemical staining is shown to be as effective as an expert consultation in resolving difficult cases. 16 Likewise, the introduction of p16 into routine staining of punch and cone biopsy specimens leads to higher interobserver agreement. 17 There is a movement to utilize p16 testing and Ki-67 staining on cytological material for the purpose of increasing interobserver agreement and to stratify patients for treatment purposes. 18 Numerous studies have examined p16 immunohistochemical staining performed on cytology specimens with a wide disparity in results (reviewed by Tsoumpou et al. 19 ). In the largest multicenter study to date, sensitivity and specificity as high as 95.1% and 71.1%, respectively, were reported for detecting HGD among ASCUS cytology specimens. 20 It should be noted that those numbers represent a process of adjudication in which discrepant cases are secondarily reviewed and reinterpreted in a group setting or with cytotechnologists, individual pathologist interpretation yielded lower sensitivity and specificity. While there is a range of sensitivity and specificity between studies to date, within individual studies the performance characteristics so far seem Fig. 3. Endocervical cells showing sporadic Ki-67 expression (red chromogen), 360. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] independent of age. 21 Efforts have been made to stratify risk based on the percentage of positively staining cells. 22 There are some technical considerations of note in the interpretation of the CINtec PLUS immunohistochemical stain that are worthy of mention. First, the interpretation of a positive result requires a nucleus stained with a red chromogen and a cytoplasm stained with brown chromogen. The background stain is hematoxylin. Others have rightly noted that the hematoxylin counterstain is different from the Papanicolaou stain. 23 We agree with the assertion that there is a subtle difference in the chromatin appearance, likely due to different bluing procedures among different hematoxylins. Second, interpretation is not a matter of positive or negative (the case in the majority of surgical pathology immunohistochemistry); rather, thoughtful evaluation of cell morphology is required to exclude squamous metaplasia, tubal metaplasia, and endocervical cells that may stain individually for p16 or Ki-67. Cells with rigid cell borders easily recognized as squamous metaplasia may show p16 positivity (Fig. 2). Groups of endocervical cells inherently show focal positive staining for Ki-67 (Fig. 3). These patterns are noted by the manufacturer, and are not to be interpreted as positive. Only dual staining cells, marked by brown and red chromogens, are to be interpreted as positive. Thus, interpretation of this immunostain requires the examiner to frequently turn to higher power to exclude normal cells that stain for an individual chromogen only. For those specimens with abundant metaplasia or a significant endocervical component, it was time consuming to 38 Diagnostic Cytopathology, Vol 41, No 1

5 CINtec PLUS SENSITIVITY AND SPECIFICITY Fig. 4. Acute inflammation expressing Ki-67 (red chromogen), 340. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] find the rare atypical cell that had been readily identified on Papanicolaou test. On one such slide, at its extreme, it took some 8 min to interpret the immunohistochemical stain. Scoring systems have been proposed and evaluated to assure that positive staining is only assessed within dysplastic cells. 21,24 In addition, acute inflammation frequently showed Ki-67 nuclear expression, causing the microscopic field of view to be filled with red chromogen reactivity (Fig. 4). Finally, the interpretation of CINtec PLUS system on SurePath specimens may have some added difficulties. Previous studies have been carried out predominantly on ThinPrep 1 preparations which have a larger button, more widely spaced cells, and are a true monolayer. SurePath slides with their smaller button have a tendency toward more three dimensional groups. The three dimensionality is present in both squamous and endocervical cells. These three dimensional clusters obscured stained cells and hindered interpretation necessitating high power examination across multiple focal planes to resolve the location (cytoplasmic or nuclear) and character (individual or dual) of staining (Figs. 5a and b). The sensitivity and specificity of CINtec PLUS seen in our study are lower than that demonstrated by other studies that have utilized a process of adjudication, but our results are roughly comparable to individual pathologist interpretation without adjudication within those same studies. Overall, the combined specificity gained by the performance of CINtec PLUS and HR-HPV testing is an improvement over current practice. The increased specificity of this test, besides simply creating another triage option, could conceivably lead to a reduction in colposcopic procedures. HR-HPV testing has been found to be a cost-effective ancillary test in analysis performed on data derived from the ALTS trial. 25 Fig. 5. a: Three dimensional cluster of endocervical cells common to SurePath preparations. Only individual staining of Ki-67 is present. Though this would be interpreted as negative, the three dimensional nature of the cluster could conceivably obscure dysplastic cells, 340. b: Three dimensional clusters of squamous cells common to SurePath preparations. These clusters may pose difficulties in interpreting the dual cytoplasmic and nuclear features of the immunohistochemical stain, 360. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] The cost-effectiveness of the CINtec PLUS system has yet to be demonstrated within the literature. Our study is far too small for such an analysis. However, it is clear that there would be an immediate procedural charge for immunohistochemical staining performed on a liquidbased preparation. The cost benefit of this ancillary test lies in avoiding colposcopy which requires both an evaluation and management charge for the patient s office visit along with the associated procedural cost of colposcopy with or without biopsy. Though more difficult to calculate, the time and discomfort of the patient is saved, is the biggest argument for increasing specificity. In conclusion, our study, though admittedly small, demonstrates an improvement in specificity above HR-HPV testing alone, and also reviews some potential difficulties Diagnostic Cytopathology, Vol 41, No 1 39

6 EDGERTON ET AL. encountered in the interpretation of this immunohistochemical stain. References 1. Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002;287: Wright TC, Jr, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D consensus guidelines for the management of women with abnormal cervical cancer screening tests. Am J Obstet Gynecol 2007;197: Edwards BK, Ward E, Kohler BA, et al. Annual report to the nation on the status of cancer, , featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates. Cancer 2010;116: Rust G, Satcher D, Fryer GE, Levine RS, Blumenthal DS. Triangulating on success: Innovation, public health, medical care, and cause-specific US mortality rates over a half century ( ). Am J Public Health 2010;100 (Suppl 1):S95 S Henley SJ, King JB, German RR, Richardson LC, Plescia M. Surveillance of screening-detected cancers (colon and rectum, breast, and cervix) United States, MMWR Surveill Summ 2010;59: Stoler MH, Schiffman M. Interobserver reproducibility of cervical cytologic and histologic interpretations: Realistic estimates from the ASCUS-LSIL Triage Study, JAMA 2001;285: van Oortmarssen GJ, Habbema JD. Duration of preclinical cervical cancer and reduction in incidence of invasive cancer following negative pap smears. Int J Epidemiol 1995;24: Franco EL, Villa LL, Sobrinho JP, et al. Epidemiology of acquisition and clearance of cervical human papillomavirus infection in women from a high-risk area for cervical cancer. J Infect Dis 1999; 180: Ho GY, Bierman R, Beardsley L, Chang CJ, Burk RD. Natural history of cervicovaginal papillomavirus infection in young women. N Engl J Med 1998;338: Nasiell K, Nasiell M, Vaclavinkova V. Behavior of moderate cervical dysplasia during long-term follow-up. Obstet Gynecol 1983;61: Mantovani F, Banks L. The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene 2001;20: Munger K, Phelps WC, Bubb V, Howley PM, Schlegel R. The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol 1989;63: Khleif SN, DeGregori J, Yee CL, et al. Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity. Proc Natl Acad Sci USA 1996;93: Key G, Becker MH, Baron B, et al. New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cdna containing three 62 base pair repetitive elements encoding for the Ki-67 epitope. Lab Invest 1993;68: Reuschenbach M, Clad A, von Knebel Doeberitz C, et al. Performance of p16ink4a-cytology. HPV mrna, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results, Gynecol Oncol 2010;119: Dijkstra MG, Heideman DA, de Roy SC, et al. p16(ink4a) immunostaining as an alternative to histology review for reliable grading of cervical intraepithelial lesions. J Clin Pathol 2010;63: Bergeron C, Ordi J, Schmidt D, Trunk MJ, Keller T, Ridder R. Conjunctive p16ink4a testing significantly increases accuracy in diagnosing high-grade cervical intraepithelial neoplasia. Am J Clin Pathol 2010;133: Wentzensen N, Bergeron C, Cas F, Vinokurova S, von Knebel Doeberitz M. Triage of women with ASCUS and LSIL cytology: Use of qualitative assessment of p16ink4a positive cells to identify patients with high-grade cervical intraepithelial neoplasia. Cancer 2007;111: Tsoumpou I, Arbyn M, Kyrgiou M, et al. p16(ink4a) immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis. Cancer Treat Rev 2009;35: Denton KJ, Bergeron C, Klement P, Trunk MJ, Keller T, Ridder R. The sensitivity and specificity of p16(ink4a) cytology vs HPV testing for detecting high-grade cervical disease in the triage of ASC-US and LSIL pap cytology results. Am J Clin Pathol 2010; 134: Szarewski A, Ambroisine L, Cadman L, et al. Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears. Cancer Epidemiol Biomarkers Prev 2008;17: Carozzi F, Confortini M, Dalla Palma P, et al. Use of p16-ink4a overexpression to increase the specificity of human papillomavirus testing: A nested substudy of the NTCC randomised controlled trial. Lancet Oncol 2008;9: Meyer JL, Hanlon DW, Andersen BT, Rasmussen OF, Bisgaard K. Evaluation of p16ink4a expression in ThinPrep cervical specimens with the CINtec p16ink4a assay: Correlation with biopsy followup results. Cancer 2007;111: Wentzensen N, Bergeron C, Cas F, Eschenbach D, Vinokurova S, von Knebel Doeberitz M. Evaluation of a nuclear score for p16ink4a-stained cervical squamous cells in liquid-based cytology samples. Cancer 2005;105: Kulasingam SL, Kim JJ, Lawrence WF, et al. Cost-effectiveness analysis based on the atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesion Triage Study (ALTS). J Natl Cancer Inst 2006;98: Diagnostic Cytopathology, Vol 41, No 1